dc.contributor.advisor | Becker, Heiko C. Prof. Dr. | de |
dc.contributor.author | Wijesekara, Kolitha Bandara | de |
dc.date.accessioned | 2013-01-31T07:57:58Z | de |
dc.date.available | 2013-01-31T07:57:58Z | de |
dc.date.issued | 2007-08-15 | de |
dc.identifier.uri | http://hdl.handle.net/11858/00-1735-0000-000D-F22A-E | de |
dc.identifier.uri | http://dx.doi.org/10.53846/goediss-3607 | |
dc.description.abstract | Raps (Brassica napus L.) hat eine
weltweite Bedeutung als Ölpflanze, aber auch als Quelle für
proteinreiche Futtermittel. Gentechnische Veränderungen sind durch
eine Agrobacterium-vermittelte Transformation grundsätzlich
möglich. Routinemäßig werden für die Transformation von Raps
diploide Pflanzenteile, meist Hypokotyle, verwendet. Aus der
Transformation von diploiden Zellen entstehen aber nur hemizygote
Transformanten, und für die Herstellung von homozygot
transformierten Pflanzen sind mehrfache Selbstungen oder der
Einsatz der Doppelt-Haploiden-Technik erforderlich. Bei einer
Transformation von haploiden Zellen oder Geweben wäre es dagegen
möglich, nach Colchizinbehandlung in einem Schritt homozygote
Transformanten zu erzeugen. Daher wurden in dieser Arbeit
Möglichkeiten zur Transformation von haploiden Pflanzenteilen mit
Hilfe von Agrobacterium untersucht. Durch Optimierung des
Transformationsprotokolls gelang es, Transformationsraten zwischen
1,8 und 2,9 % zu erreichen. Die erfolgreiche Transformation konnte
über PCR nachgewiesen werden. Daher kann die Transformation von in
vitro vermehrten haploiden Pflanzen als schnelle Methode zur
Erzeugung homozygoter transgener Pflanzen bei Raps empfohlen
werden. Ein Problem im Rapsanbau ist die relativ große
Lageranfälligkeit. In diesem Zusammenhang ist eine Verkürzung der
Wuchshöhe von großem Interesse. Daher wurden Möglichkeiten zur
Reduktion der Wuchshöhe durch Überexpression des Phytochrome B Gens
aus Arabidopsis untersucht. Transformierte Pflanzen zeigten einen
höheren Chlorophyllgehalt und eine reduzierte Appikaldominanz. In
der T2 Generation waren homozygot transgene Pflanzen um 28 % in der
Wuchshöhe reduziert. Diese Pflanzen zeigten allerdings auch einen
sehr stark verzögerten Blühzeitpunkt, so dass eine unmittelbare
züchterische Nutzung dieses transgenen Ansatzes schwierig
erscheint. | de |
dc.format.mimetype | application/pdf | de |
dc.language.iso | eng | de |
dc.rights.uri | http://creativecommons.org/licenses/by-nc-nd/3.0/ | de |
dc.title | Development of a haploid transformation system and overexpression of Phytochrome B gene in Brassica napus L. | de |
dc.type | doctoralThesis | de |
dc.title.translated | Entwicklung eines haploiden transformationssystem und überexpression des Phytochrom B gene bei Brassica napus L. | de |
dc.contributor.referee | Becker, Heiko C. Prof. Dr. | de |
dc.date.examination | 2007-07-19 | de |
dc.subject.dnb | 580 Pflanzen (Botanik) | de |
dc.subject.gok | YEA 900 | de |
dc.description.abstracteng | Oilseed rape (Brassica napus L.) is a
renowned crop worldwide for its edible oil as well as a source of
protein animal feed. Agrobacterium-mediated transformation has
proved to work efficient for genetic engineering of this crop. The
majority of successful transformation systems hitherto described
for B. napus use diploid plant materials such as hypocotyls,
cotyledons, and cotyledonary petioles. When diploid plant materials
were used, first generation transgenic plants (T1) would be
hemizygous for the transformed gene(s). Consequently, much time and
labour has to be invested to produce homozygous transgenic plants
by repeated selfing or by applying the doubled haploid technology.
The use of haploid cells and tissue explants could provide an
alternative approach for efficient transformation of oilseed rape,
since haploid transgenic plants will become homozygous in one step
after colchicine treatment. This study reports the potential of
using leaf and petiole explants of haploid plants propagated in
vitro for an efficient Agrobacterium-mediated gene transfer.
Compared to a standard protocol for hypocotyl segments,
significantly higher callus production (P=0.05) with leaf explants
was achieved when cultured in callus induction medium CIM-L (MS
basal medium supplemented with 1 mg/l BAP, 0.1 mg/l NAA, 0.1 mg/l
GA3, 5 mg/l AgNO3 and solidified with 5 g/l
Agarose). For petiole explants CIM-P medium (MS basal supplemented
with 2 mg/l BAP, 0.01 mg/l picloram, 5 mg/l AgNO3 and
solidified with 5 g/l Agarose) had a significant positive effect
(P=0.05) on callus production. Highest shoot regeneration was
achieved in DKW medium supplemented with 1 mg/l BAP, 0.1 mg/l NAA,
0.1 mg/l GA3, and 5 mg/l AgNO3 and solidified
with 5 g/l Agarose. Analysis of the ploidy showed that 96% of
plantlets regenerated from leaf and 92% from petiole explants
remained haploid. Using Agrobacterium tumefaciens strain AGL0,
which carried the binary plasmid pAK-UGI 9-1, a transformation
efficiency of 1.8% and 2.9% was achieved for leaf and petiole
explants, respectively. Presence of GUS gene in regenerated
plantlets was confirmed by PCR. Results indicate that
transformation of leaf and petiole explants derived from in vitro
propagated haploid plants provides a suitable alternative for
generation of homozygous transgenic plants in rapeseed. Lodging
could be a serious problem in oilseed Brassica napus. Lodging could
be decreased by reducing plant height. This study investigated the
possibility of reducing plant height by overexpression of A.
thaliana PHYB gene in B. napus plants. Ectopic expression of
Arabidopsis PHYB cDNA under the control of constitutive CaMV35S
promoter in Brassica napus caused pleiotropic effects such as
decreased apical dominance, a higher number of small leaves and
increased leaf chlorophyll content. A strictly light-dependent
short hypocotyl phenotype was observed with transgenic seedlings
under in vitro culture conditions. Supposed homozygous transgenic
plants in the segregating population (T2) exhibited substantially
modified plant architecture with reduced apical dominance.
Beginning of flowering in these plants was significantly delayed
when compared with control plants. At maturity, supposed homozygous
transgenic plants remained 28% shorter than the corresponding
controls. Even though PHYB overexpressing plants resulted in
reduced plant height, which could reduce lodging under field
conditions, extremely delayed flowering make these plants difficult
to fit into a rapeseed breeding program. | de |
dc.contributor.coReferee | Karlovsky, Petr Prof. Dr. | de |
dc.contributor.thirdReferee | Maass, Brigitte PD Dr. | de |
dc.subject.topic | Agricultural Sciences | de |
dc.subject.eng | Brassica napus | de |
dc.subject.eng | Agrobacterium-mediated transformation | de |
dc.subject.eng | haploid transgenic plants | de |
dc.subject.eng | phytochrome B | de |
dc.subject.eng | overexpression | de |
dc.subject.bk | 48.58 | de |
dc.identifier.urn | urn:nbn:de:gbv:7-webdoc-1555-2 | de |
dc.identifier.purl | webdoc-1555 | de |
dc.identifier.ppn | 554127830 | de |